T3 Uptake polarization fluoroimmunoassay

ABSTRACT

A T 3  uptake polarization fluoroimmunoassay where a sample is assayed by contacting a serum sample with T 3  to form a solution, contacting the solution with a separating agent and incubating the same, separating T 3  bound to the separating agent from the serum sample, contacting the T 3  bound to the separating agent with an antibody against T 3 , incubating and separating the agent-anti-body combination, separating free anti-body and contacting the free antibody with fluorescent labeled T 3  and fluorometrically measuring the fluorescence polarization.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a T₃ uptake immunoassay and, in particular, to a T₃ uptake polarization fluoroimmunoassay.

2. Description of the Prior Art

As early as 1939, Treverrow (1) reported that hormonal iodine compounds such as thyroxine (T₄) constitute the major portion of the total serum iodine. Furthermore, these iodinated organic compounds could be distinguished from serum inorganic iodide because they are bound to serum protein. Since iodine constitutes 65 percent by weight of the T₄ molecule, serum protein-bound iodine (PBI) was employed as an index of serum T₄. This indirect measurement of serum T₄ was believed to be a good indicator of the thyrometabolic status of an individual (2,3,4). The normal range of PBI values was found to be 4-8 ug/100 ml; values below 4 ug/100 ml were consistent with hypothyroidism whereas values above 8 ug/100 ml were indicative of thyrotoxicosis (hyperthyroidism).

A major pitfall of the PBI test is its inherent lack of specificity since high levels of inorganic iodide, radio-opaque dyes and certain drugs give abnormally high values. In 1964, Murphy et al. (5) introduced their competitive protein binding analysis (CPB) for serum T₄ which solved most of the nonspecificity problems associated with the PBI test. Due to the fact that CPB tests for serum T₄ also required an extraction of the T₄ from the remainder of the serum components, recovery variability has lead to problems both in accuracy and precision.

Recently, radioimmunoassay (RIA) has become the method of choice for measuring serum T₄ (6). The RIA technique can be run directly on serum without extraction and therefore yields a simple and yet highly specific test. In general, results from RIA are from 5 to 25 percent higher than those from CPB tests.

Although the direct measurement of serum T₄ is not influenced by exogenous iodine, the value obtained will be influenced by the level of the circulating T₄ binding proteins. A number of states which are totally unrelated to thyroid disease may cause abnormal serum levels of T₄. Changes in the serum level of circulating T₄ binding proteins may cause the serum T₄ level to be high or low even in the presence of normal thyroid function. Although the primary protein involved is thyroxine binding globulin (TBG), both thyroxine binding prealbumin (TBPA) and albumin also bind T₄. Normally T₄ is distributed as follows: 65% on TBG, 25% on TBPA, and 10% on albumin (7). In general, changes in TBG concentrations correlate much better with anomalies in thyroid function tests, such as the PBI or total T₄ than do changes in TBPA (8).

Although the most accurate method to measure TBG concentrations involves the electrophoretic method of Orsorio et al. (9), the technique is too cumbersome for routine use. The method of choice which has been used for this purpose is one of the many variations of the triiodothyronine (T₃) uptake test. Hamolsky et al. (10) first performed this type of test. All T₃ uptake tests are designed to assess the unsaturated binding capacity of serum proteins most notably TBG. The test is based on the fact that TBG binds T₃ less firmly than T₄ and therefore should not upset the equilibrium set-up between T₄ and TBG and, further, T₃ is not normally bound to TBPA.

In the T₃ uptake test an equilibrium is developed between the patient's serum, added labeled T₃ and an inert exogenous binder (separating agent) of the labeled T₃. One must add a sufficient amount of labeled T₃ to saturate the binding sites on the TBG after which the labeled T₃ that is unbound is adsorbed by the separating agent and the separating agent bound labeled T₃ is counted. Therefore, when the endogenous T₄ level is increased, as in hyperthyroidism, serum TBG is relatively saturated and the T₃ uptake will be high. Conversely in the hypothyroid state where T₄ output is low, the labeled exogenous T₃ will bind to the relatively unsaturated TBG yielding a low T₃ uptake.

Presently, radioassay is a method of choice for performing a T₃ uptake test. However, this T₃ uptake test has severe disadvantages in that radiolabeled T₃, e.g., ¹²⁵ I-labeled T₃, is relatively unstable and radioassay kits are restricted by regulations such as those governing the transport, handling, and storage of radioactive materials.

It would therefore be very desirable to develop an alternative T₃ uptake test which retains the advantages present in the radioassay technique but which overcomes the two problems inherent therein, namely the relative instability of radiolabeled T₃ and the restrictions placed upon radioassay kits.

Although the bases for various fluoroimmunoassays (11) such as fluorescent polarization (12-15), enhancement fluoroimmunoassay (16), fluorescent excitation transfer immunoassays (17), and fluorescent immonuabsorbent tests (18) have been described and demonstrated, to data, other than a fluorescent polarization assay of gentamicin (19) and dilantin (20) none of the above methodologies have been applied in a practical assay of proven clinical utility. For example, in the case of enhancement fluoroimmunoassays (16), it is reported that the variable levels of intrinsic serum fluorescence present an obstacle to the assay of patient samples.

Bibliography

1. Trevorrow, V., J. Biol. Chem., 127:737 (1939).

2. De Mowbray, R. R., et al., Lancet, 2:511 (1952).

3. Sunderman, F. W., et al., Amer. J. Clin. Path., 24:885 (1954).

4. Dailey, M. E., et al., New Engl. J. Med., 254 (19):907 (1956).

5. Murphy, B. E. P., et al., J. Clin. Endocrinol., 24:187 (1964).

6. Chopra, I. J., J. Clin. Endocrinol., 34:938 (1972).

7. Robbins, J., et al.: Hormones in Blood, Gray, C. H., et al., eds., Academic Press, London, 2nd Ed., 1:430 and 447 (1967).

8. Thomas, J. A., et al.: Hormone Assays and Their Clinical Application, Loraine, J. A., et al., eds., Churchill Livingston, New York, 4th Ed., Vh. 12 (1976).

9. Osorio, C., et al., Clin. Sci., 21:355 (1961).

10. Hamolsky et al., J. Clin. Endocrinol., 17:33 (1957).

11. O'Donnell, Analytical Chemistry, 50 (5):189R (1978).

12. Dandliker, W. D. et al., Immunochem., 7:799 (1970).

13. Dandliker, W. D., et al., Research Communications and Chemical Pathology and Pharmacology, 18 (1):147 (1977).

14. Levinson, J. A., Endocrinology, 99:1129 (1976).

15. Dandliker, W. D., et al., Immunochemistry, 1:165 (1964).

16. Smith, D. S., Fubs Letters, 77 (1):25 (1977).

17. Ullman, E. F., et al., J. of Biol. Chem., 251:4172 (1976).

18. Aalberse, R. C., Clin. Chem. Acta, 48:109 (1973).

19. Watson, R. A. A., et al., Clin. Chem. Acta, 73:51 (1976).

20. McGregor, A. R., et al., Clin. Chim. Acta, 83:161 (1978).

SUMMARY OF THE INVENTION

In accordance with the present invention an improved T₃ uptake test is provided which retains the advantages present in the radioassay technique but which overcomes the two problems inherent therein. The T₃ uptake test of the instant invention is a T₃ uptake polarization fluoroimmunoassay. More particularly, the protocol of the T₃ uptake fluoroimmunoassay of this invention comprises:

(a) contacting a sample to be assayed with T₃ to thereby form a solution;

(b) contacting an aliquot of the solution with a separating agent;

(c) incubating the aliquot-separating agent combination;

(d) separating T₃ bound to separating agent from serum bound T₃ ;

(e) contacting the T₃ bound to separating agent with an antibody against T₃, said antibody having a higher affinity for T₃ than the affinity of the separating agent for T₃ ;

(f) incubating the separating agent-antibody combination;

(g) separating free antibody and T₃ bound antibody from the separating agent;

(h) contacting the free antibody with fluorescent labeled T₃ ; and

(i) fluorometrically measuring the fluorescent polarization in the mixture of step (h). The value obtained by the fluorometric measurement of step (i) is directly proportional to the serum uptake of the added T₃ and indirectly proportional to the conventional T₃ uptake value as measured via radioassay T₃ uptake methodologies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a correlation analysis of a radioassay T₃ uptake procedure versus a ratio of the peak rate of the control serum to the peak rate of the sample as obtained in a polarization fluoroimmunoassay uptake procedure within the scope of this invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Samples which can be assayed by the instant invention for T₃ uptake include biological fluids having either an unknown T₃ uptake (e.g., a patient's sample) or biological fluids having a known T₃ uptake (i.e., standards). Typical biological fluids include, but are not limited to, plasma and serum. Serum is the preferred biological fluid employed in the analysis of the instant invention.

Any separating agent whose affinity for T₃ is less than the affinity of serum proteins (most notably TBG) for T₃ can be used in the instant invention. Typical separating agents include, but are not limited to, bead-formed dextran gel and agarose. Preferably, the separating agent employed in the instant invention is bead-formed dextran gel.

Any antibody having a higher affinity for T₃ than the affinity of the particular separating agent for T₃ can be used in the instant invention. Typical T₃ antibodies include, but are not limited to, purified sheep, rabbit, and goat IgG fraction containing T₃ antibody.

Any suitable fluorescent label can be used to label the T₃. Fluorescent labels include, but are not limited to, fluorescien and rhodamine. Fluoroescien is the preferred fluorescent label.

The incubation time of step (c) is not critical and can be any convenient period of time. Preferably, this incubation step is conducted for about 5 to about 60 minutes, preferably for about 15 minutes.

Similarly, the incubation procedure of step (f) is not critical and can also be conducted for any convenient period of time. Preferably, this incubation step is conducted for about 0.5 to about 2 hours, and more preferably for about 1 hour.

The decrease in polarization fluorescence caused by contacting the free antibody with the fluorescent labeled T₃ can be measured by any well known fluorometric techniques via either an endpoint or kinetic methodology. Preferably, a kinetic fluorometric technique is employed in the assay of this invention.

Preferably, after step (g) the free antibody is contacted with a non-fluorescent surfactant. Non-fluorescent surfactants include, but are not limited to, octylphenoxy polyethoxy ethanol and polyoxyethylene sorbitan monolaurate. Octylphenoxy polyethoxy ethanol is the preferred non-fluorescent surfactant.

The problems of non-specific bonding and of variable background fluorescence caused by serum are overcome by step (d) of the procedure of the instant invention when the T₃ which is bound to the separating agent is removed from the serum.

The following examples are provided for the purposes of illustration only and are not intended to be limitations on the disclosed invention.

EXAMPLE 1 T₃ Uptake Polarization Fluoroimmunoassay A. Reagents

1. Purified sheep IgG fraction containing T₃ antibody diluted 1/800 in B-2 buffer (0.075 M barbital buffer comprising, per liter of water, 15.6 gm sodium barbital, 2.0 gm sodium EDTA ((ethylenedinitrilo)tetraacetic acid), and 9.0 gm sodium chloride) containing 1 g/l gelatin.

2. Fluorescein labeled T₃ (FT₃) at a concentration of 250 pg T₃ /50 μl in B-2 buffer containing 0.1 mg/ml insulin.

3. T₃ solution at a concentration of 2.5 ng/200 μl in B-2 buffer containing 1 mg/ml bovine serum albumin (BSA) and 1 mg/ml gelatin.

4. Octylphenoxy polyethoxy ethanol (0.01%) in B-2 buffer.

B. Preparation of Columns

1. Individual columns were prepared employing 5.0 ml aliquots obtained from a suspension of 25 gms of bead-formed dextran gel (sold under the trademark Sephadex G25 (Fine)) in about 500 ml of saline. Each column was fitted with a disc prepared from Whatman No. 2 brand filter paper and washed with 4.0 ml of B-2 buffer.

C. SamplePreparation

1. In clean borosilicate glass tubes 150 μl B-2 buffer, 50 μl sample, and 200 μl of T₃ solution were mixed. Next, 300 μl aliquots, of this mixture, were delivered on top of the individual columns, allowed to penetrate into the gel bed, and then incubated therein for about 15 minutes.

2. After 4.0 ml of B-2 buffer had been washed through each column, 300 μl of T₃ antibody was delivered on top of the individual columns and allowed to penetrate into the gel bed and then incubated therein for about 1 hour.

3. Finally, each column was washed with 1.5 ml of B-2 buffer and the eluants were collected in clean borosilicate tubes. Following the addition of 100 μl of B-2 buffer containing 0.01% octylphenoxy polyethoxy ethanol, the individual aliquots were vortexed.

D. Polarization fluoroimmunoassay of Free Antibody to T₃

1. Aliquots (500 μl) from each tube were reacted with fluorecein labeled T₃ and the resulting fluoroescein polarization of the aliquot was measured.

a. The data obtained are inversely related to the T₃ uptake of each sample. The data obtained from this experiment are set forth in Table I.

EXAMPLE 2 Radioassay T₃ Uptake

In order to evaluate the efficacy of the T₃ uptake polarization immunoassay of the instant invention, a correlation study was performed employing the following radioassay T₃ uptake procedure set forth in Tri-Tab T₃ Uptake Diagnostic Kit, Nuclear-Medical Laboratories, Inc., Dallas, Texas.

The data obtained via the above radioassay T₃ uptake protocol are set forth in Table I.

                                      TABLE I                                      __________________________________________________________________________           Radioassay                 Polarization                                   Sample                                                                               ##STR1##  Peak Rate (H)                                                                          Mean                                                                               VariationofCoeff.                                                                   ##STR2##                                     __________________________________________________________________________                    879                                                             1     0.68     902      890 1.83%                                                                               0.58                                                         840                                                             2     0.70     819      830 1.79%                                                                               0.62                                                         811                                                             3     0.76     823      817 1.04%                                                                               0.63                                                         806                                                             Hyland A                                                                             0.76     776      791 2.68%                                                                               0.65                                                         764                                                             4     0.77     799      782 3.17%                                                                               0.66                                                         757                                                             5     0.85     744      750 1.22%                                                                               0.69                                                         601                                                             6     0.93     632      616 3.56%                                                                               0.84                                                         580                                                             7     0.98     600      590 2.40%                                                                               0.88                                                         524                                                             Hyland B                                                                             1.01     510      517 1.91%                                                                               1.00                                                         519                                                             8     1.05     482      500 5.23%                                                                               1.03                                                         448                                                             9     1.07     447      448 0.16%                                                                               1.15                                                         381                                                             10    1.20     369      375 2.26%                                                                               1.38                                                         304                                                             11    1.30     314      309 2.29%                                                                               1.67                                                         340                                                             12    1.32     343      342 0.62%                                                                               1.52                                                         304                                                             Hyland C                                                                             1.35     324      314 4.50%                                                                               1.65                                                         317                                                             13    1.35     326      322 1.98%                                                                               1.65                                                         306                                                             14    1.42     305      306 0.23%                                                                               1.69                                                         296                                                             15    1.58     300      298 0.95%                                                                               1.74                                          __________________________________________________________________________

FIG. 1 is a plot of the ratio of the counts per minute (cpm) for a sample being assayed to the cpm of the standardizing sample being assayed by a radioassay T₃ uptake procedure vs. the ratio of the peak rate for Hyland B to the peak rate for the sample being assayed by the instant invention. The plot shown in FIG. 1 demonstrates excellent correlation (r=0.98) between the assay of the instant invention and a conventional T₃ uptake radioassay. In addition, the average coefficient of variation for the data set forth in Table I is 2.01% which is comparable to the average coefficient of variation (2.33%) of the radioassay T₃ uptake procedure employed in Example 5.

Since the ratio of cpm Hyland B sample to cpm of the standardizing sample is 1.01 (see Table I), it can be concluded that the cpm for the Hyland B sample is substantially identical to the cpm of the standardizing sample employed in the radioassay T₃ uptake procedure.

As noted above, the value obtained by the fluorometric measurement of the instant invention is indirectly proportional to T₃ uptake values as measured via conventional radioassay T₃ uptake procedures. This relation can be mathematically shown as ##EQU1## Since cpm Hyland B≃cpm standardizing sample, one can used Hyland B as the standardizing sample in the polarization fluoroimmunoassay under the assumption that cpm Hyland B is proportional to H Hyland B. Therefore, ##EQU2## The fact that the assumption that cpm Hyland B is proportional to H Hyland B is correct is evidenced by the excellent correlation (r=0.98) between these two methodologies.

It should be noted that FIG. 1 graphically depicts the fact that the methodology of the instant invention yields a larger normal and hypothyroid region than obtainable with the radioassay T₃ uptake procedure employed in Example 5. This fact tends to make the methodology of the instant invention more accurate than that of the prior art.

Based upon this disclosure, many other modifications and ramifications will naturally suggest themselves to those skilled in the art. These are intended to be comprehended as within the scope of this invention. 

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
 1. A T₃ uptake polarization fluoroimmunoassay procedure comprising:(a) contacting a serum sample to be assayed with T₃ to thereby form a solution, the amount of said T₃ being sufficient to assess the unsaturated binding capacity of serum proteins; (b) contacting an aliquot of said solution with a separating agent; (c) incubating said aliquot-separating agent combination; (d) separating T₃ bound to separating agent from said serum and thereby simultaneously removing nonspecific serum effects as well as background fluorescence; (e) contacting said T₃ bound separating agent with an antibody against T₃, said antibody having a higher affinity for T₃ than the affinity of said separating agent for T₃ and the amount of said antibody being sufficient to remove at least substantially all of said T₃ from said separating agent; (f) incubating said separating agent-antibody combination; (g) separating free antibody and T₃ bound antibody from said separating agent; (h) contacting said free antibody with fluorescent labeled T₃ ; and (i) fluorometrically measuring the fluorescence polarization in said mixture of step (h);wherein said measurement is inversely proportional to the T₃ uptake of the sample being assayed.
 2. The assay of claim 1 wherein said separating agent is selected from a group consisting of bead-formed dextran gel and agarose.
 3. The assay of claim 1 wherein said fluorescent labeled T₃ is selected from a group consisting of fluorescein labeled T₃ and rhodamine labeled T₃.
 4. The assay of claim 3 wherein after step (g) a non-fluorescent surfactant is contacted with said free antibody.
 5. The assay of claim 4 wherein:(a) said separating agent is selected from a group consisting of bead-formed dextran gel and agarose; (b) said fluorescent labeled T₃ is selected from a group consisting of fluorescein labeled T₃ and rhodamine labeled T₃ ; and (c) after step (g) said free antibody is contacted with a non-fluorescent surfactant.
 6. The assay of claim 5 wherein said separating agent is bead-formed dextran gel.
 7. The assay of claim 5 wherein said fluorescent labeled T₃ is fluorescein labeled T₃.
 8. The assay of claim 5 wherein said non-fluorescent surfactant is selected from a group consisting of oxylphenoxy polyethoxy ethanol and polyoxyethelene sorbitan monolaurate.
 9. The assay of claim 5 wherein:(a) said separating agent is bead-formed dextran gel; (b) said fluorescent labeled T₃ is fluorescein labeled T₃ ; and (c) said non-fluorescent surfactant is selected from a group consisting of oxylphenoxy polyethoxy ethanol and polyoxyethelene soribatan monolaurate.
 10. The assay of claim 9 wherein said non-fluorescent surfactant is octylphenoxy polyethoxy ethanol.
 11. The assay of any one of claims 1-10 wherein said fluorometric measurement is a kinetic fluorometric measurement. 